Intermediate filaments (IF) are the ubiquitous constituents of the cytoskeletons of eukaryote cells. They consist of five different types, of which the most numerous and complex are the type I and type II keratins that are widely expressed in epithelia. We are interested in not only the structure, function and expression of keratin IF of human skin and their roles in keratinopathy diseases, but also of the related IF of other cell types in order to understand their roles in biology. Ongoing structural studies While the roles of the keratins in many genetic diseases are now well understood, further structural studies are necessary to develop rational approaches to therapy. We have initiated a major study in collaboration with other investigators in Switzerland, Germany and New Zealand to solve the three-dimensional structure of vimentin IF by use of Xray crystallographic techniques. These IF have been chosen because: (a) they are homopolymeric, and therefore likely to be somewhat simpler to solve; and (b) they have a very high sequence homology with keratin IF, and thus many of the structural principles adduced for vimentin should be applicable to keratin IF. To date, we have solved the structure of the last 35 residues of the 2B rod domain segment, which encompasses the helix-termination motif, and the likely trigger motif required for successful IF assembly. Work is now in progress on numerous other constructs have been made which cumulatively cover the entire rod domain portion of vimentin. The organization of molecules in trichocyte keratin IF We have previously demonstrated by detailed cross-linking experiments that pairs of epidermal keratin molecules are aligned in three modes termed A11, A22 and A12. When assimilated into IF, pairs of molecules in the same axial row adopt a fourth mode termed ACN, in which the end of one molecule overlaps the beginning of the adjacent molecule by about 1 nm. Interestingly, almost all known keratinopathy mutations/substitutions reside in this overlap window. We also have shown that the molecules of type III IF adopt the same basic four modes, but the alignments of the former three are slightly offset. This adequately explains why type III and types I/II keratin chains cannot and do not coassemble in vivo or in vitro. We have now re-examined these questions in trichocyte IF, and have found that the alignments for reduced trichocyte keratin IF are exactly the same as for cytokeratin IF. However, when trichocyte keratins are oxidized, a series of disulfide bonds form that shift the A11 alignment mode, with the net result of a 1 nm gap between the end and beginning of adjacent molecules. Interestingly, several of the disulfide bonds occurred between head domain and rod domain cysteines, which suggests that the head domain is intimately involved in the molecular shifts and is essential for the maintenance of stability of the trichocyte IF structure. The only way this could occur is if the head domain sequences fold back over the rod domain. This may explain why head domain sequences are essential for successful IF assembly. Accordingly, we have made two series of constructs. In the first, various portions of the head domains of the Type I or Type II chains have been deleted and these will be used for assembly experiments. In this way, we hope to define which head domain sequences are necessary for each hierarchical stage of IF assembly. In the second set of constructs, we have replaced one at a time each arginine residue on head domain sequences with a lysine and then used the proteins for protein chemical cross-linking experiments. In this way, we will be able to define precisely how the head domain interacts with the rod domain segments, and the net effect this may have on IF assembly. Furthermore, we have initiated experiments on the structures of trichocyte (hair) keratins. Several constructs encompassing rod domain and head + rod domain segments have been made and expressed. After purification, the physico-chemical properties of each protein construct will be tested to assure that it forms uniform homopolymeric complexes in solution, and if successful, the proteins will then be used for crystallization trials. The organization of molecules in vimentin IF Assuming that vimentin head domain sequences also interact with rod domain in IF assembly and stabilization, we have made similar types of deletion and arginine-to-lysine substitution constructs. Together, both sets of experiment may point to common and/or different mechanisms by which different Types of IF are maintained in cells. Structure of the end domains We have discovered a novel mutation in the gene encoding keratin 1 which causes a frameshift and change in the coding sequence of the tail domain. In the wildtype protein, such sequences are enriched in glycines, but in the mutant, the frame has been changed to alanine-rich instead. The net result is a severe case of ichthyosis hystrix Curth-Macklin. It appears the abnormal keratin tail allows IF to form, but severely interferes with keratin IF function. In particular, the distribution of loricrin to the cell periphery is prevented, resulting in a severe barrier function defect. This is not only the first mutation reported for a keratin end domain in disease, but now allows for new insights into the structure and function of the tail domains. To test this model in a live animal context, we have made a ?knockin? keratin 1 gene construct in which the equivalent mutation in the mouse keratin 1 chain has been introduced. Animal work is now in progress. Searches for high molecular weight IF associated proteins When type III vimentin IF are passaged through cycles of assembly and disassembly in vitro, certain high molecular weight proteins always co-cycle. We have purified one of these from BHK-21 fibroblasts, and shown that it is the type VI IF protein nestin. This protein can be incorporated in small amounts into vimentin IF and its long tail is thought to serve as an interfilamentous spacer to separate individual IF and affect supramolecular organization in cells. We believe that functionally similar molecules should exist for keratin IF. Keratin IF in epithelia usually occur in tonofibril bundles containing 100s of individual IF loosely separated by 1-3 filament diameters from each other. Ongoing experiments are designed to look for keratin IF organizing or spacer proteins in epithelial cells. In preliminary experiments, we used mass spectroscopy to look for novel high molecular weight proteins in extracts of keratin IF from cultured immortalized epithelial cells. One interesting protein that was identified is epiplakin, a new member of the plakin family of cytolinkers. To being to test whether this protein could serve the bundling function, we have made a new antibody in rabbits and will test this using immuno-fluorescence and immuno-precipitation experiments.